PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with a five-year survival rate of less than 8%. These grim statistics owe, in part, to the profound resistance of PDA to current therapies. More effective treatment strategies would offer tremendous benefit to PDA patients. Emerging evidence suggests that epigenetic and genetic heterogeneity exist in both the neoplastic and non-neoplastic compartments of pancreatic tumors, and that this heterogeneity may impact response to treatment. Our own studies have identified two subpopulations of cancer-associated fibroblasts (CAFs) present within the PDA microenvironment, a cancer-proximal population that expresses myofibroblast markers and a cancer-distal population that expresses inflammatory markers such as the cytokine IL-6. However, the full complement of cell populations present within the PDA tumors and the mechanisms through which each population might impact tumor biology remain unclear. Tackling these questions requires models that accurately recapitulate human PDA. Patient-derived xenografts (PDXs), organoid cultures, organoid-stromal co-cultures, and organoid xenografts have emerged as ?next-generation? models that better mimic the complex interactions present in the PDA microenvironment. Yet, the extent to which each model preserves the diverse populations found in human tumors remains unclear. To address this question, we propose to generate a series of matching organoid and xenograft models which we will use to characterize and perturb subpopulations of neoplastic cells and CAFs. We will generate and characterize five sets of paired organoid culture and PDX models, where pairs are derived the same de-identified patient tumor specimens. In addition, we will develop a new model for pancreatic cancer in which patient-derived PDA organoids are delivered to mice in situ via injection into the main pancreatic duct, better recapitulating the developmental trajectory of human PDA than other xenograft approaches. We will use single-cell RNA and DNA sequencing as well as barcoded organoid lines to characterize and perturb the neoplastic and CAF populations present in our models. To study to role of IL-6 in PDA more directly, we will use biochemical and genetic strategies to perturb CAF-mediated IL-6 signalling in our models. Among these, we will make use of a murine model engineered to express human IL-6 as a host for our PDA xenografts, restoring the ability for IL-6 generated in the stroma to signal to the transplanted neoplastic cells. Ultimately, our studies will shed light on the distinct subpopulations of neoplastic cells and CAFs present in the PDA microenvironment and help to identify which of those subpopulations function to promote the aggressive traits characteristic of PDA tumors. The knowledge gained from our studies will provide valuable insights into the nature of the tumor microenvironment. Such insights should inform novel ideas for strategies to effectively treat PDA, a clinical goal with urgent unmet need.